TRANSPLANTATION AND CELLULAR ENGINEERING Successful short-term cryopreservation of volume-reduced cord blood units in a cryogenic mechanical freezer: effects on cell recovery, viability, and clonogenic potential Ioannis Anagnostakis, Andreas C. Papassavas, Efstathios Michalopoulos, Theofanis Chatzistamatiou, Sofia Andriopoulou, Athanassios Tsakris, and Catherine Stavropoulos-Giokas BACKGROUND: Cord blood (CB) units are stored from weeks to years in liquid- or vapor-phase nitrogen until they are used for transplantation. We examined the effects of cryostorage in a mechanical freezer at -150°C on critical quality control variables of CB collec- tions to investigate the possible use of mechanical freezers at -150°C as an alternative to storage in liquid- (or vapor-) phase nitrogen. STUDY DESIGN AND METHODS: A total of 105 CB units were thawed and washed at different time inter- vals (6, 12, 24, and 36 months). For every thawed CB unit, samples were removed and cell enumeration (total nucleated cells [TNCs], mononuclear cells [MNCs], CD34+, CD133+) was performed. In addition, viability was obtained with the use of flow cytometry, and recov- eries were calculated. Also, total absolute colony- forming unit counts were performed and progenitor cell recoveries were studied by clonogenic assays. RESULTS: Significant differences (p < 0.05) were observed in certain variables (TNCs, MNC numbers, viability) when they were examined in relation with time intervals, while others (CD34+, CD133+) were relatively insensitive (p = NS) to the duration of time interval the CB units were kept in cryostorage condition. CONCLUSIONS: The data presented suggest that cryopreservation of CB units in a mechanical freezer at -150°C may represent an alternative cryostorage condi- tion for CB cryopreservation. U mbilical cord blood (UCB) is accepted as a viable alternative to bone marrow (BM) for the treatment of hematologic disorders as a source of hematopoietic stem cells (HSCs). 1-10 In the years after the first human cord blood (CB) trans- plantation, CB banks have evolved and finally became a critically important factor for the success of UCB trans- plantation. 11 Criteria of donor choice have been identified with cell dose to be the most widely used factor. 1,12-15 With more than 400,000 CB units cryopreserved for long periods in more than 100 CB registries and at least 21 countries involved, 11,16 it becomes critically important that a given unit being considered for transplantation still has viable hematopoietic progenitors. Although HSCs have been successfully recovered after up to two decades of cryostorage 17,18 several other studies have noted a gradual decline in HSC quality and functional characteristics with increasing periods of storage. 19-21 Cryopreservation of hematopoietic progenitor cells using liquid nitrogen requires complex equipment and highly trained staff and is expensive. Also, difficulty ABBREVIATIONS: BM = bone marrow; CB = cord blood; HSC(s) = hematopoietic stem cell(s); TNC(s) = total nucleated cell(s); UCB = umbilical cord blood. From the Hellenic Cord Blood Bank, Foundation for Biomedical Research, Academy of Athens; the Department of Immunology and National Tissue Typing Center, “G. Gennimatas” General Hospital; and the Department of Microbiology, Medical School, University of Athens, Athens, Greece. Address reprint requests to: Efstathios Michalopoulos, PhD, Hellenic Cord Blood Bank (HCBB), Foundation for Biomedical Research, Academy of Athens, 4 Sorranou Efessiou Street, 11527 Athens, Greece; e-mail: smichal@bioacademy.gr. Received for publication August 20, 2012; revision received March 28, 2013, and accepted April 2, 2013. doi: 10.1111/trf.12239 TRANSFUSION 2014;54:211-223. Volume 54, January 2014 TRANSFUSION 211